New technologies for DNA sequencing and extracting human DNA sequences such as Next Generation Sequencing (NGS) were gradually developed to achieve higher throughput for extraction of genomic information at a lower cost. NGS technologies consist of three main stages: template preparation, base calling (which is mostly based on imaging) and processing. In the template preparation stage, DNA fragments are first randomly broken to smaller fragments, and such fragments are attached to solid surface of a template platform. Following this step, two different approaches may be used for preparing templates: (a) amplification of a single molecule and (b) single molecule preparation. Because detection of the signal corresponding to a single molecule can be challenging, amplification of a single molecule is the step commonly adopted in many practical NGS methods, such as in Illumina technology. Subsequent to the template preparation stage, an observed signal must be detected for the base calling. The detected signal may be the temperature, pH, or intensity of fluorescence photons. After detecting nucleotides, generated reads must be processed at the last stage. The processing stage may consist of alignment or assembly of fragments methods which can also exploit an existing reference genome.
In order to resolve repeats on the genome or to determine Structural Variations (SVs) between target and reference genomes, paired-end sequencing is being developed. In paired-end sequencing, larger fragments of DNA samples can be used. After a bio-chemical process, two paired sequences are read by sequencer machine from the fragment with a known insert size between them. In the traditional scheme, the paired-end sequencing process is performed for each one of two sequences independently. Consequently, after bounding these larger fragments to the substrate and amplification step, an enzyme is used to detach reverse stands from the substrate. A sequencing procedure is then started for the forward strands. Following the sequencing of the forward stands, DNA polymerase is again added to the solution in order to construct reverse stands. An enzyme is also added to detach the forward stands and the sequencing of the reverse strands is carried out. It can be understood that current paired-end sequencing methods require an extended period of time and involve the consumption of large amounts of material.
Hence, there is a need for a method for sequencing both DNA strands (i.e., the forward strand and the reverse strand) simultaneously. In such a scheme, only half of the traditional method's materials would be consumed. In addition, the time needed for the sequencing process to occur may be halved relative to traditional paired-end sequencing methods.